High-resolution X-ray structure of Gln143Asn manganese superoxide dismutase captures multiple hydrogen...

Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide ({\rm O_{2}^{{\bullet}-}) into hydrogen peroxide (H₂O₂) and molecular oxygen (O₂), serving as a key defence against oxidative damage. Despite extensive studies, the full structural characterization of H₂O₂-binding sites in MnSOD remains largely unexplored. Previous H₂O₂-soaked MnSOD structures have identified two distinct H₂O₂-binding sites: one directly ligated to the catalytic manganese (LIG position) and another at the active-site gateway (PEO position) between the second-shell residues Tyr34 and His30. In this study, a kinetically impaired Gln143Asn MnSOD variant is used to trap and explore additional H₂O₂-binding sites beyond the second-shell solvent gate. In the wild-type enzyme, Gln143 mediates proton transfers with the manganese-bound solvent (WAT1) to drive redox cycling of the metal, which is necessary for effective {\rm O_{2}^{{\bullet}-} dismutation. Substitution with Asn stalls catalysis because the increased distance from WAT1 disrupts critical proton-coupled electron-transfer (PCET) events, and the redox cycling of the active-site metal is impaired. This, in turn, stalls the electrostatic cycling of positive charge on the enzyme surface and enhances the likelihood of trapping transient H₂O₂-bound states in this variant. The results reveal several H₂O₂ molecules leading up to the active site, in addition to the canonical LIG and PEO positions.